Micro Biology Lab Packet

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Ex 3-7: The Gram Stain

Pages 195-202

Today we will do 3 things:

Observe the 2 streak plates and assess the broth culture and slants from Tuesday for growth patterns

Gram stain some reference organisms

Observe our environmental plates on dissecting scope for Colony Morphology (Ex. 2-2)

Tools to characterize and identify microorganisms

  • Microscopic morphology
  • Colony morphology
  • Culture characteristics
  • Biochemical activities

  • Each of these is determined by an organism’s genes, and will be consistent for a bacterial species or isolate

Culture Characteristics in Broth

  • Surface:

Ring, pellicle, none

  • Sediment:

present, absent

  • Broth Clarity:

Uniform turbidity, flocculent *, clear

  • Amount of growth:

0 to +3 (none/slight/moderate/abundant)

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Growth Patterns in Broth

  • pellicle*growth on surface
  • flocculent (clumps)
  • uniform turbidity
  • sediment

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Serratia marcescens. Gram negative, short bacilli that produce mucoid colonies which have entire margins and umbonate elevation. Note that there are both red and white colonies present on this plate. Some strains of S. marcescens produce the red pigment prodigiosin in response to incubation at 30o C, but do not do so at 37o C. This is an example of temperature-regulated phenotypic expression.

Broth: turbid appearance of the broth and the red color present in both the sediment and pellicle.

Slant: white to red color, smooth, moist growth

Micrococcus luteus. Circular, pinhead colonies which are convex with entire margins. This gram positive coccus produces a bright yellow, non-diffusable pigment.

Broth: produced granular and pellicle growth

Slant: abundant, opaque, grayish white, smooth, flat, slimy, and moist.

Escherichia coli. This gram negative, coccobacillus forms shiny, low convex, mucoid colonies which have entire margins and are slightly raised. Older colonies often have a darker center.

Broth: turbid growth with a deposit which disperse upon shaking.

Slant: off-white in color and showed a spreading growth pattern: usually it appeared as a cloudy film over the whole surface of the slant


Staphylococcus saprophyticus A Gram-positive, cocci forms slightly convex colonies that are opaque, smooth, circular, gray-white, white to cream colonies.

Broth: uniform turbidity with a fine to slight mucoid deposit.

Slant:

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Culture Characteristics on a Slant

  • Slants are useful for keeping stock cultures of bacteria
  • Growth occurs on slant
  • The butt* allows for diffusion of nutrients to the organisms and diffusion of waste products away from organisms
  • Observe pattern of growth on the slant:

  • Note any other unique/obvious characteristics – e.g., color

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  • Purpose:
  • To determine appearance of growth on various media

  • This is consistent for an organism and is useful to characterize and identify organisms
  • Genetically determined, yet environmentally influenced
  • Compare organisms to see if they’re the same or different
  • Can tell us about their properties, such as the conditions/media at which they grow

3) Colony Morphology/Culture Characteristics

Colony Morphology/Culture Characteristics

7 Characteristics of Colony Morphology:

  • Shape
  • Margin
  • Elevation
  • Size
  • Color
  • Texture
  • Optical Properties

  • Shape

  • Elevation

  • Margin

Colony Characteristics (p. 64)

  • Size – measure in mm (or a comment)
  • Color – describe the color (specifically)
  • Texture – typically refers to the colony surface or its texture when probed with a loop
  • Optical Properties – how it transmits light (opaque, translucent, transparent)

Dissecting scopes are useful to observe colonies

Colony Characteristics

Summary: Characteristics to observe

On a plate

(as an isolated colony)

Colony Morphology

  • Shape
  • Margin
  • Elevation
  • Size
  • Color
  • Texture
  • Optical properties

On a slant

Growth on slants

Pattern of growth

Anything else that’s obvious or unique

In a broth

Growth in Broth

Surface

Sediment

Broth

Amount of growth

Streak Plate

Last time you created a streak plate from a mixed broth culture (Namely, E. coli, M. luteus, and Serratia marcescens)

A good streak plate:

  • Is appropriately labeled
  • Has an appropriate pattern
    for streak lines
  • Uses the entire plate
  • Has well-isolated colonies
  • Is free of contamination

Did you obtain individual colonies?

Observe & critique your streak plate

To get more well-isolated colonies:

  • Use a small amount of initial bacterial inoculum (in first quadrant).
  • Remember to flame loop after each quadrant.
  • Don’t go back into previous quadrant so much (2 or 3 pass throughs).
  • Keep streak lines moving in forward direction.
  • Use entire plate.

Types of Stains used by Microbiologists

  • Simple Stains – a single dye is used to add contrast, so microbes can be viewed microscopically
  • Positive stain
  • Negative stain
  • Differential Stains
  • Gram stain
  • Acid-fast stain
  • Special Structure Stains
  • Capsule stain
  • Spore stain
  • Flagellar stain

X

  • Differential stains detect differences between organisms
  • The Gram stain differentiates bacteria into two groups: gram-positive and gram-negative, based on differences in cell wall structure.
  • Gram-positive bacteria tend to be killed more easily by antibiotics like penicillin.
  • Gram-negative bacteria can be more resistant to antibiotics.

Differential Stains: Gram Stain

Will react in a crystal violet dye and will stain dark purple or violet.

Cell wall is unique because it is composed of several peptidoglycan layers (multilayered) making it thicker.

Presence of techoic acids

Lacks an LPS (lipopolysachharide) content, periplasmic space and outer membrane making this group low in both lipoprotein and lipid composition.

Gram Positive

Normally does not react to a crystal violet dye, stains pinkish red [decolorized in accepting safranin (a counterstain)]

Thinner peptidoglycan cell wall layer because it is just made of one single layer as opposed to gram positive’s multi-layered wall

Lacks teichoic acids, but has a high content of LPS

Has both a periplasmic space and an outer membrane

Has a high amount of lipoprotein content as well as lipids.

Gram Negative

Gram Stain
Start with an air-dried, heat-fixed smear.

primary stain mordant decolorizer counterstain

  • Crystal violet-iodine complexes form in cell
  • Gram-positive bacteria
  • Alcohol dehydrates & condenses thick peptidoglycan layer
  • Crystal violet-iodine complexes do not leave

  • Gram-negative bacteria
  • Alcohol dissolves outer membrane (LPS) and leaves holes in thin peptidoglycan layer
  • Crystal violet-iodine washes out

Gram Stain Mechanism

Differential Stains: Gram Stain

For every Gram stain, observe & record:

Gram reaction

cell shape

cell arrangement

size (if you can)

Gram Stain – Common Problems

  • Smears are too thick
  • Decolorizing too little or too much
  • Old cells and dead cells tend to stain Gram negative
  • Because the cell wall is not intact
  • Should always use fresh cultures

Demonstration of a Gram stain

  • Air-dried, heat-fixed smear
  • Remember: small amount of organism spread out in a small drop of water

  • Gram stain procedure – p 200
  • Decolorization is the most crucial step

Until run-off is clear

page 200

Expectations: Each student should:

Observe & critique your streak plates

Prepare & Gram stain 1 slide

  • Slide: Three smears on slide:

Gram Pos., Mixed, and Gram Neg.

  • Gram stain procedure – p 200

  • Observe with oil immersion objective (100 X)

  • Observe simple stained slides from last lab and colony morphology

S. Sapro

Or M. luteus

S. marcescens

Or E. coli

Mixed

coccus bacillus spirillum

Bacterial Shape

  • Singly
  • Pairs: diplococci, diplobacilli
  • Clusters: staphylococci
  • Chains: streptococci, streptobacilli

Arrangement

For Next Lab:

Read:

  • Ex 3.9 Capsule Stain
  • Ex 3.10 Endospore Stain
  • Ex 3.12 Flagella Stain

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